A Study on the Effect of Dithiothreitol and Beta-mercaptoethanol on the Production of Lysosomal Aggregates

Protein aggregates have attracted much attention in scientific communities in recent years. Human lysozyme (HuL) is an amyloidogenic protein. Since hen egg white lysozyme (HEWL) and HuL have a high degree of structural and sequential homology, it has been used as a model of protein aggregation in this study. In this project we studied the effects of reducing agents such as dithiothreitol and β-mercaptoethanol on the aggregation of lysozyme by the use of thioflavin T (ThT) fluorescence, circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). First we tried to set up the preliminary conditions for HEWL amyloid aggregation which was incubation of HEWL (2 mg/mL) for 24 h at 54°C and pH 2.0. We included dithiothreitol and β-mercaptoethanol in the aggregation mixtures and incubated for 1, 2, 4 and 8 hours at above stated conditions. At the third stage, the effect of dithiothreitol and β-mercaptoethanol on preformed lysosomal aggregates, in the presence of guanidine thiocyanate was examined and it became clear that both of them decreased the fluorescence emission, and resulted in a decrease in the lysosomal aggregates. Circular dichroism spectroscopy results showed that dithiothreitol and β-mercaptoethanol reduced the intensity of fluorescence in the region of beta sheet peak in comparison with the control sample, indicating a reduction in the density of aggregates. Also, AFM images showed that in the presence of dithiothreitol and β-mercaptoethanol, the number and thickness of amyloid fibrils decreased. It was found that disulfide bond reducing agents could reduce lysozyme aggregation. These results would presumably help to understand better the phenomenon of aggregation from a physico-chemical point of view. It is also suggested that these kinds of reducing agents could also be used in the mechanistic studies related to the design of novel drugs against amyloidogenic diseases caused by the accumulation of peptides and proteins.